Identification of chromatin‐related protein interactions using protein microarrays

Abstract

Dynamic structural changes in chromatin are mediated by protein interactions that modulate multiple cellular processes including replication, transcription, recombination and DNA repair. Complexes that recognize chromatin are defined by several distinct groups of proteins that either directly modify histones or interact with histone-DNA complexes. A protein microarray format was used to analyze the interaction of various DNA repair proteins with chromatin components. We applied proteins, antibodies and DNA to functionalized glass slides and interrogated the slides with our proteins of interest to identify novel protein-protein interactions for proteins involved in DNA double-strand break repair. Here we demonstrate that the DNA repair protein RAD51B, and not its cognate partner RAD51C, interacts with histones and not nucleosomes. Nucleosome-specific interactions were demonstrated with the recently identified SWI/SNF protein, SMARCAL1. Unique RAD51B-histone interactions were corroborated using Far Western analysis. This is the first demonstration of an interaction between RAD51B and histone proteins that may be important for the successful repair of DNA double-strand breaks.

Advances in chromatin remodeling and human disease

“…Consistent with its putative role in chromatin remodeling, the SMARCAL1 protein binds nucleosomes but not isolated histone proteins [29]. Although the function of SMARCAL1 has not been defined biochemically, clinical findings suggest that SMARCAL1 may regulate a subset of genes involved in the proliferation of affected tissues [30].…”

Validation and Quality Control of Protein Microarray-based Analytical Methods

“…For microarrays in their current state of development, many groups have utilized replicate analyses of a given sample. Strategies include duplicate [51,55] triplicate [53], quadruplicate [56,57], and up to 10 replicate analyses of the sample on the same array [58][59][60], and replicates to assess spatial artifacts [7]. Other replication strategies include adjacent duplicates on two different arrays [61].…”

Gene Clusters, Molecular Evolution and Disease: A Speculation

“…Both clinical and cell culture studies suggest that functional SMARCAL1 protein is required for the proliferation of many of the affected tissues [114][115][116]. Consistent with its role as an annealing DNA helicase [117], the SMARCAL1 protein binds nucleosomes and has DNA-dependent, RNA-independent, ATPase activity [118,119]. The SMARCAL1 protein is predominantly localized to the open chromatin and loss of functional SMARCAL1 apparently causes cell autonomous disease by altering chromatin helical torsion [117,120].…”